Biocontrol of Orchid-pathogenic Mold, Phytophthora palmivora, by Antifungal Proteins from Pseudomonas aeruginosa RS1

Figures & data

Figure 1. Inhibition of P. palmivora by P. aeruginosa RS1 on a V8 agar plate (A) and control growth of the mold without the bacteria (B). Microscopic observation (400×) of the hyphae of P. palmivora growing with (C) and without (D) P. aeruginosa RS1.

Figure 2. Inhibition percentage against P. palmivora and absorbance at OD_660 nm of P. aeruginosa RS1 in LB medium (pH 7) at 37 °C, at time points measured every 3 h, 24 h.

Table 1. Percentage of inhibition by culture filtrate from P. aeruginosa RS1 at different variable factors.

Variable factors	Conditions	% Inhibition
Media	NB, pH7	29.03 ± 2.63
	LB, pH7	53.33 ± 2.72
	TSB, pH7	13.33 ± 3.85
pH	LB, pH6	48.57 ± 2.33
	LB, pH7	52.63 ± 2.15
	LB, pH8	32.35 ± 2.40
	LB, pH9	22.58 ± 2.63
Temperature	LB, pH7, 30 °C	41.94 ± 2.63
	LB, pH7, 37 °C	54.84 ± 3.72
	LB, pH7, 40 °C	35.48 ± 2.63
Ammonium sulfate	0–40%	0.00 ± 2.55
	40–80%	100.00 ± 0.00
	80–100%	0.00 ± 2.55

Figure 3. Chromatogram from DEAE-cellulose column chromatography indicates the absorbance at OD_280 nm of the proteins eluting from the column using gradient concentrations of NaCl.

Figure 4. Inhibition of P. palmivora by eluted proteins from DEAE-cellulose column chromatography. P. palmivora grew on a V8 agar plate: control without protein (A), with protein fraction 9–10 (B), and with protein fraction 33–34.

Figure 5. SDS-PAGE of partial purification of the antifungal proteins from the ammonium sulfate precipitation and anion exchange chromatography steps. The labels 9–10 and 33–34 refer to the proteins from DEAE fractions 9–10 and 33–34, respectively.

Table 2. Summary for purification of the antifungal protein from P. aeruginosa RS1.

Sample	Volume (ml)	Protein (ng/ml)	Total protein (mg)	Activity (AU/ml)	Specific activity (AU/mg protein)	Total activity (AU)	Purification (fold)
Culture filtrate	5000.00	72.00	360.00	1.60	22.22	8000.00	1.00
(NH4)2SO4 40–80%	24.00	718.00	17.232	16.00	22.28	384.00	1.00
DEAE #9–10	4.20	8.00	0.0336	2.00	250.00	8.40	11.25
DEAE #33–34	4.00	108.00	0.432	4.00	37.04	16.00	1.67

Table 3. Identification of each protein band from anion exchange chromatography fractions 9–10 and 33–34.

Sample	Log (e)	Protein coverage	No. of unique peptide found	Molecular weight (kDa)	Accession No.	Protein	Organism
Fraction 9–10, #1	−737.5	65%	64	55.6	gi ǀ 218889435	Catalase	Pseudomonas aeruginosa LESB58
Fraction 9–10, #2	−1881.8	53%	145	53.7	gi ǀ 218890000	Protease	Pseudomonas aeruginosa LESB58
Fraction 9–10, #3	−1349.0	40%	99	48.2	gi ǀ 218889496	Piv gene product (protease IV)	Pseudomonas aeruginosa LESB58
Fraction 33–34, #4	−1648.1	86%	128	41.9	gi ǀ 218893181	Chitin binding protein	Pseudomonas aeruginosa LESB58
Fraction 33–34, #5	−1881.8	53%	145	53.7	gi ǀ 218890000	Protease	Pseudomonas aeruginosa LESB58
Fraction 33–34, #6	−864.7	33%	68	45.5	gi ǀ 218892171	Protease	Pseudomonas aeruginosa LESB58

Figure 6. Morphology of P. palmivora under scanning electron microscope (2000×) after incubation for 2 days without (A) and with the purified antifungal protein from DEAE-cellulose column chromatography at fractions 9–10 (B) and 33–34 (C).